Synthesis, antimicrobial and cytotoxic activities, and structure–activity relationships of gypsogenin derivatives against human cancer cells

Accepted Manuscript Synthesis, antimicrobial and cytotoxic activities, and structure-activity relationships of gypsogenin derivatives against human cancer cells Safiye Emirdağ-Öztürk , Tamer Karayıldırım , Aysun Çapcı-Karagöz , Özgen AlankuşÇalışkan , Ali Özmen , Esin Poyrazoğlu-Çoban PII:

S0223-5234(14)00516-9

DOI:

10.1016/j.ejmech.2014.05.084

Reference:

EJMECH 7042

To appear in:

European Journal of Medicinal Chemistry

Received Date: 5 February 2014 Revised Date:

15 May 2014

Accepted Date: 31 May 2014

Please cite this article as: S. Emirdağ-Öztürk, T. Karayıldırım, A. Çapcı-Karagöz, Ö. Alankuş-Çalışkan, A. Özmen, E. Poyrazoğlu-Çoban, Synthesis, antimicrobial and cytotoxic activities, and structure-activity relationships of gypsogenin derivatives against human cancer cells, European Journal of Medicinal Chemistry (2014), doi: 10.1016/j.ejmech.2014.05.084. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

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ACCEPTED MANUSCRIPT

ACCEPTED MANUSCRIPT

Synthesis, antimicrobial and cytotoxic activities, and structure-activity relationships of gypsogenin derivatives against human cancer cells

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Safiye Emirdağ-Öztürk a,*,Tamer Karayıldırım a, Aysun Çapcı-Karagöz b,Özgen Alankuş-Çalışkan a, Ali Özmen c, and Esin Poyrazoğlu-Çoban c a

Chemistry Department, Faculty of Science, Ege University, Bornova, Izmir 35100, Turkey Institute of Organic Chemistry I, University of Erlangen-Nuremberg, Henkestrasse 42, 91054 Erlangen, Germany Biology Department, Adnan Menderes University, Aydin 09010, Turkey

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Abstract

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A series of gypsogenin (1) derivatives (1a-i) was synthesized in good yields, and the derivatives’ structures were 1 13 established using UV, IR, H NMR, C NMR, and LCMS spectroscopic data. Among the tested compounds, 1a, 1b, 1d, 1e, and gypsogenin (1) showed antimicrobial activities against Bacillus subtilis and Bacillus thrungiensis, with inhibition zones of 10–14 mm. In addition, compounds 1b, 1d, and 1e showed antimicrobial activities against Bacillus cereus, with inhibition zones of 9–14 mm. Using six human cancer cell lines in vitro, the cytotoxic activities of all tested compounds were determined by calculating the IC50 values. Doxorubicin and paclitaxel were used as controls. Among the tested compounds, 1a, 1c, and 1d had inhibitory effects with IC50 values of 3.9 µM (HL-60 cells), 5.15 µM (MCF-7 cells), and 5.978 µM (HL-60), respectively. To determine the type of cell death, Hoechst 33258 (HO) and propidium iodide (PI) double staining was used. Especially, gypsogenin (1) and compound 1a triggered the apoptotic mechanism at a concentration of 20 µM. Thus, gypsogenin (1) and compounds 1a, 1c, and 1d possess varying degrees of biological activities and can be considered as potential antitumor agents. Keywords: Gypsogenin; saponin; Gypsophila; cytotoxic; apoptosis

1. Introduction

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Saponins, which are detected in a number of plant families, are glycosides with a polycyclic aglycone and a sugar moiety. Sugars can attach to aglycone, which is also called sapogenin, at one or two different positions; thus, they are named monodesmosidic and bidesmosidic saponins, respectively [1-3]. These secondary metabolites have hydrophilic and hydrophobic sides; hence, they have surface activity, and saponin-containing plants are used as soap. Saponins have various structure-dependent biological activities such as glucosidase inhibiting [4], antiviral [5, 6], anti-inflammatory [7], spermicidal [8], hypocholesterolemic [9], antitumor [10, 11], anticarcinogenic [12], and antioxidant activities [13]. Moreover, saponins have been evaluated against cancer cells for anticancer activity [14, 15]. Triterpene saponins can be found in many plant species [16-19], and several recent studies have reported on saponins produced from Gypsophila

species [20-22]. Some saponins from Gypsophila have shown a variety of biological activities including anticarcinogenic [23], immunostimulatory [24], and cytotoxic activities [25]. Gypsophila accumulate gypsogenin aglycone with sugar chains, which has been attributed to various biological properties. For example, these compounds have exhibited inhibitory activity [26] and have shown significant growth inhibition in in vitro cultures [27]. In addition, some of them have shown very high activity against different human cancer cell lines [28, 29]. Thus, there is strong evidence that gypsogenin has anticancer activity. Gypsogenin aglycone is found at high concentrations in Gypsophila [30]; therefore, it can be obtained with ease [31]. In this study, nine new gypsogenin derivatives (1a-i) were synthesized from gypsogenin aglycone (1). In addition, they were evaluated for their antibacterial and antifungal activities as well as cytotoxic activities against six different human cancer cell cultures.

* Corresponding author: S.Emirdağ-Öztürk.,Tel.: (+) 90 232 311 2371 Fax: (+) 90 232 388 8264; E-mail: [email protected], [email protected]

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ACCEPTED MANUSCRIPT Usually, this rection is preferred to protect the carboxyl

2.1. Chemistry

Compound

R1

R2

Gypsogenin

-OH

-COH

1a

-OH

-CH=NOH

1b

-OCOCH3

-CHO

1c

-OH

-CHO

1d

-OCOCH3

1e

R3

Yield (%)

-COOH

-

-COOH

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Table 1 Structures and yields of gypsogenin (1) and its derivatives (compounds 1a-i).

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Nine gypsogenin derivatives (1a-i) were synthesized by a series of reactions as outlined in Scheme 1. The starting material, gypsogenin (1), was obtained from the commercially available Gypsophila arrostii root extract, and its isolation has been explained in our previous work [32]. All compounds were obtained in good yields, as shown in Table 1.

group of aglycone [33-35]. These intermediates were then reacted with thiosemicarbazide in 1:1 methanol: water to yield compounds 1e and 1g, respectively. Compounds 1b and 1c were converted into compounds 1d and 1f, respectively, by treatment with hydroxylamine hydrochloride at room temperature. Acetylation of compound 1c at C-3 gave compound 1h, which was further reacted with hydroxyl hydrochloride to afford compound 1i. The nine synthesized compounds were established 1 13 1 by IR, UV, H NMR, C NMR, and LCMS analyses. H NMR spectroscopy was especially useful to confirm the structures. For example, for compound 1a, the signal for the aldehyde proton at δH 10.75 was converted to an 1 oxime group at δH 7.88. The H NMR spectrum also showed a doublet of doublets at δH 4.17 (J=16.0 and 5.2 Hz) attributed to H-3. 1 In the H NMR for compound 1b, the proton signal of H-3 at δH 5.51 was observed instead of at δH 5.20 ppm. This conversion was also seen for compound 1h. The 1 H NMR spectrum of compound 1c showed the extra 13 proton signal of OCH2-C6H5 at δH 5.16 ppm. In the C NMR, the signal of OCH2-C6H5 appeared at δC 66.46 ppm. For compound 1e, the signals for –N=CH and NH2C=S could be seen at δH 7.51 and δH 9.73, 13 respectively. These signals were also evident by the C NMR spectrum showing –N=CH and NH2C=S at δC 153.76 and δC 181.00, respectively. These spectral data are summarized in Tables 2 and 3 and the Supplementary data (Figure 1).

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2. Results and discussion

97.8

-COOCH2C6H5

54.6

-CH=NOH

-COOH

97.2

-OCOCH3

-CH=NNHCSNH2

-COOH

45.8

1f

-OH

-CH=NOH

-COOCH2C6H5

88.3

1g

-OH

-CH=NNHCSNH2

-COOCH2C6H5

56.9

1h

-OCOCH3

-CHO

-COOCH2C6H5

97.7

1i

-OCOCH3

-CH=NOH

-COOCH2C6H5

91.2

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-COOH

Gypsogenin (1) was treated with hydroxylamine hydrochloride and sodium acetate in 3:1 acetonitrile: water at room temperature to provide compound 1a. The intermediate compounds 1b and 1c were synthesized by substitution reactions involving acetylation at C-3 and benzylation at C-28, respectively.

2.2. Biological activity 2.2.1. Antimicrobial activity The antimicrobial activities of gypsogenin (1) and its nine new derivatives were analyzed by the disc diffusion method [36] and minimum inhibitory concentration (MIC) determination [37]. The antimicrobial activities of these compounds were evaluated against different strains of Gram-negative bacteria (Escherichia coli ATCC 25922, Salmonella typhimurium ATCC 14028, Klebsiella pneumonia ATCC 13882, Pseudomonas aeruginosa ATCC 35032, and Proteus vulgaris ATCC 33420), Gram-positive bacteria (Micrococcus luteus ATCC 9341, Stapylococcus aureus ATCC 25923, Stapylococcus epidermidis ATCC 12228, Bacilllus cereus ATCC 11778, Bacillus subtilis ATCC 6633, Bacillus thrungiensis, and Entereococcus faecalis ATCC 29212), and yeast (Candida utilis ATCC 9950, Candida albicans ATCC 10231, Candida glabrata, Candida trophicalis, and Saccharomyces cerevisiae ATCC 9763). The results of the in vitro antimicrobial activities of the tested compounds, along with known antibiotic and antifungal reagents for comparison, are reported as inhibition zone (mm) and MIC values and are listed in Tables 4 and 5, respectively.

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1d

1e

1a

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1b

1c

1f

1h

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1g

1i

Scheme 1. Reagents and conditions: (a) hydroxylamine hydrochloride (NH2OH·HCl), sodium acetate, 3:1 acetonitrile: water, rt; (b) acetic anhydride, pyridine, rt; (c) benzyl bromide, triethylamine, reflux; (d) thiosemicarbazide, 1:1 MeOH: water, reflux.

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ACCEPTED MANUSCRIPT Table 2 1 H NMR data of gypsogenin (1) and compounds 1a-i (400 MHz, δ ppm, in pyridine). Position

Gypsogenin (1)a,b [32] 1aa

1ba

1ca

Compound 1da

1ea

1fa

1ga

1ha

1ia 1.56 (m), 1.01 (m)

2.66 (m), 2.17 (m)

1.92 (m), 1.36 (m)

1.84 (m), 1.36 (m)

1.91 (m), 1.25 (m)

1.82 (m), 1.34 (m)

1.76 (m), 1.20 (m)

1.66 (m), 1.05 (m)

1.69 (m), 1.04 (m)

1.67 (m), 1.00 (m)

3.23 (m), 2.26 (m)

2.40 (m), 1.47 (m)

2.40 (m), 1.22 (m)

1.89 (m), 0.08 (m)

2.02 (m), 1.06 (m)

4.36 (m)

5.44 (m)

5.53 (m)

4.02 (m)

5.22 (m)

5.16 (m)

4 5 6 7 8 9 10 11 12 13 14 15 16 17

2.58 (m) 2.61 (m), 2.29 (m) 2.59 (m), 2.31 (m) 2.89 (m) 2.98 (m), 2.46 (m) 6.62 (br s) 2.61 (m) 3.25 (m), 3.06 (m) 4.43 (dd), (J=13.6, 4.0 Hz) 2.92 (m), 2.45 (m) 2.56 (m), 2.35 (m) 3.15 (m), 2.93 (m) 10.75 (s) 2.48 (s) 2.01 (s) 2.11 (s) 2.40 (s) 2.08 (s) 2.13 (s)

1.73 (m) 1.79 (m), 1.37 (m) 1.76 (m), 1.43 (m) 2.02 (m) 2.10 (m), 1.61 (m) 5.72 (br s) 1.82 (m) 2.34 (m), 2.19 (m) 3.45 (dd), (J=18.4, 4.4 Hz) 2.05 (m), 1.57 (m) 1.70 (m), 1.45 (m) 2.23 (m), 2.05 (m) 9.91 (s) 1.66 (s) 1.06 (s) 1.23 (s) 1.52 (s) 1.22 (s) 1.24 (s)

1.71 (m) 1.76 (m), 1.49 (m) 1.72 (m), 1.51 (m) 1.99 (m) 2.21 (m), 1.61 (m) 5.76 (br s) 1.79 (m) 2.42 (m), 1.77 (m) 3.58 (dd), (J=17.6, 4.4 Hz) 2.11 (m), 1.59 (m) 1.69 (m), 1.54 (m) 2.33 (m), 2.18 (m) 1.64 (s) 1.20 (s) 1.26 (s) 1.57 (s) 1.25 (s) 1.32 (s) 7.60 (s) 2.24 (s)

1.60 (m) 1.70 (m), 1.24 (m) 1.68 (m), 1.24 (m) 2.02 (m) 2.26 (m), 1.50 (m) 5.76 (br s) 1.73 (m) 2.42 (m), 2.30 (m) 3.59(dd), (J=17.6, 3.6 Hz) 2.10 (m), 1.45 (m) 1.56 (m), 1.25 (m) 2.34 (m), 2.18 (m) 1.54 (s) 1.18 (s) 1.41 (s) 1.31 (s) 1.25 (s) 1.27 (s)

2.02 (m), 1.07 (m) 3.92 (dd), (J=16.0, 5.2 Hz) 1.49 (m) 1.63 (m), 1.12 (m) 1.60 (m), 1.16 (m) 1.77 (m) 1.93 (m), 1.36 (m) 5.43 (br s) 1.67 (m) 2.05 (m), 1.95 (m) 3.17 (dd), (J=17.6, 3.6 Hz) 1.81 (m), 1.27 (m) 1.46 (m), 1.19 (m) 1.97 (m), 1.84 (m) 1.41 (s) 0.80 (s) 0.93 (s) 1.21 (s) 0.94 (s) 0.98 (s)

2.02 (m), 1.05 (m)

5.20 (m)

2.37 (m), 1.37 (m) 5.51 (dd), (J=16.8, 5.6 Hz) 1.63 (m) 1.75 (m), 1.43 (m) 1.70 (m), 1.43 (m) 2.04 (m) 2.22 (m), 1.55 (m) 5.76 (br s) 1.80 (m) 2.39 (m), 2.30 (m) 3.59 (dd), (J=18.0, 3.6 Hz) 2.11 (m), 1.51 (m) 1.61 (m), 1.45 (m) 2.33 (m), 2.19 (m) 9.79 (s) 1.58 (s) 1.15 (s) 1.27 (s) 1.47 (s) 1.25 (s) 1.32 (s)

2.33(m), 1.35 (m)

3

2.40 (m), 1.46 (m) 4.17 (dd), (J=16.0, 5.2 Hz) 1.80 (m) 1.87 (m), 1.54 (m) 1.83 (m), 1.56 (m) 2.04 (m) 2.26 (m), 1.73 (m) 5.80 (br s) 1.91 (m) 2.43 (m), 2.31 (m) 3.60 (dd), (J=18.0, 4.0 Hz) 2.12 (m), 1.63 (m) 1.78 (m), 1.56 (m) 2.35 (m), 2.19 (m) 1.75 (s) 1.25 (s) 1.30 (s) 1.58 (s) 1.27 (s) 1.32 (s) 7.88 (s)

1.45 (m) 1.55 (m), 1.14 (m) 1.53 (m), 1.16 (m) 1.81 (m) 1.88 (m), 1.23 (m) 5.42 (br s) 1.64 (m) 2.06 (m), 1.89 (m) 3.17 (dd), (J=17.6, 3.6 Hz) 1.77 (m), 1.22 (m) 1.28 (m), 1.20 (m) 1.90 (m), 1.83 (m) 9.52 (s) 1.24 (s) 0.76 (s) 0.94 (s) 1.21 (s) 0.90 (s) 0.96 (s)

1.43 (m) 1.52 (m), 1.12 (m) 1.46 (m), 1.15 (m) 1.66 (s) 1.82 (m), 1.34 (m) 5.42 (br s) 1.53 (m) 2.06 (m), 1.88 (m) 3.16 (dd) (J=17.6, 4.0 Hz) 1.77 (m), 1.25 (m) 140 (m), 1.19 (m) 1.90 (m),1.79 (m) 1.38 (s) 0.77 (s) 0.96 (s) 1.23 (s) 0.94 (s) 0.98 (s) 7.60 (s) 1.97 (s) 4.90 7.54 (d), (J=7.2 Hz) 7.45 (t), (J=8.0, 7.2 Hz) 7.37 (t), (J=8.0, 8.0 Hz)

B (C6H5) C (C6H5) D (C6H5) -CHNNH -CSNH -CSNH2 a

1.56 (m) 1.61 (m),1.14 (m) 1.58 (m), 1.16 (m) 1.72 (m) 1.80 (m),1.41 (m) 5.44 (br s) 1.64 (m) 1.96 (m), 1.83 (m) 3.17 (dd), (J=17.6, 3.6 Hz) 1.74 (m), 1.28 (m) 1.53 (m), 1.19 (m) 1.87 (m), 1.78 (m) 1.49 (s) 0.95 (s) 0.98 (s) 1.23 (s) 0.96 (s) 1.00 (s) 7.75 (s)

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2.23 (s)

2.20 (s)

5.16 7.55 (d), (J=8.0 Hz) 7.45 (t), (J=8.0, 8.0 Hz) 7.37 (t), (J=8.0, 8.0 Hz)

5.12 7.55 (d), (J=8.0 Hz) 7.44 (t), (J=8.0, 7.2 Hz) 7.37 (t), (J=7.2, 7.2 Hz)

EP

19 20 21 22 23 24 25 26 27 28 29 30 -CHNOH -CH3COO -CH2-C6H5

AC C

18

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1 2

7.51 (s) 8.60 (br s) 9.73 (br s)

5.29 7.55 (d), (J=7.6 Hz) 7.45 (t), (J=8.0, 7.2 Hz) 7.37 (t), (J=7.2, 7.2 Hz) 7.23 (s) 8.74 (br s) 9.38 (br s)

1.95 (s) 4.85 7.55 (d), (J=8.0 Hz) 7.45 (t), (J=8.0, 8.0 Hz) 7.37 (t), (J=8.0, 8.0 Hz)

Multiplicity of signals is given in parentheses: s, singlet; d, doublet; t, triplet; m, multiplet; and br, broad. Identified from the HMBC, HMQC, and NOESY spectra.

b

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Table 3 C NMR data of gypsogenin (1) and compounds 1a-i (100 MHz, δ ppm, in pyridine).

13

a

Identified from the HMBC, HMQC, and NOESY spectra.

21.11 170.41

Compound 5 38.14 28.60 76.66 52.66 47.00 20.36 33.50 40.28 48.28 37.11 26.50 122.50 145.20 42.50 31.29 24.01 46.94 42.29 46.75 32.98 36.06 33.59 12.70 16.08 17.64 24.10 180.42 34.55 23.63

6 39.06 28.34 75.57 52.26 47.39 20.52 33.12 40.33 48.48 37.20 27.44 123.20 144.43 42.40 31.13 23.99 47.32 42.28 46.42 32.55 36.07 33.45 12.67 16.33 17.61 26.41 177.50 34.30 23.74 159.72

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4 38.33 28.59 77.38 52.73 46.94 20.28 33.50 40.30 48.32 37.09 26.52 122.53 145.23 42.52 31.29 24.04 46.76 42.30 45.72 32.99 36.76 33.60 13.02 16.13 17.64 24.10 180.42 34.56 23.62 157.46 21.39 170.47

SC

3 38.85 28.31 72.01 56.57 48.03 21.33 33.09 40.35 48.23 36.46 27.40 123.06 144.42 42.41 31.14 23.99 47.30 42.27 46.43 32.82 36.16 33.45 207.53 10.02 16.05 17.55 26.38 177.46 34.30 23.33

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2 38.09 28.57 73.84 54.94 48.11 21.07 33.48 40.33 48.12 36.34 26.49 122.42 145.22 42.54 31.29 23.99 46.94 42.30 46.78 32.71 36.30 33.60 204.93 10.07 15.78 17.60 24.10 180.40 34.56 23.14

TE D

38.24, CH2 28.02, CH2 71.38, CH 56.04, qC 47.46, CH 20.97, CH2 32.78, CH2 39.81, qC 47.84, CH 35.81, qC 26.82, CH2 122.02, CH 144.64, qC 42.21, qC 30.85, CH2 23.82, CH2 46.62, qC 41.76, CH 46.22, CH2 32.32, qC 35.50, CH2 33.05, CH2 206.87, qC 9.51, CH3 15.34, CH3 17.35, CH3 25.93, CH3 179.85, qC 33.97, CH3 23.54, CH3

1 39.04 28.64 75.57 52.32 47.40 20.57 33.51 40.38 48.58 37.24 27.44 122.73 145.22 42.56 31.29 24.14 46.99 42.37 46.81 33.22 37.20 33.61 12.65 16.29 17.74 26.51 180.45 34.57 24.02 159.77

7 38.93 28.34 75.24 52.20 48.48 20.65 33.10 40.32 48.70 37.07 27.55 123.16 144.41 42.36 31.13 23.97 47.32 42.27 46.42 33.12 36.05 33.45 12.20 16.27 17.59 26.37 177.49 34.30 23.72

21.30 170.76

66.46 137.59 129.22 128.69 128.76

66.44 137.62 129.22 128.68 128.76

EP

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 -CHNOH -CH3COO -CH3COO -CH2-C6H5 A (C6H5) B (C6H5) C (C6H5) D (C6H5) -CSNH2 -CHNNH

a

Gypsogenin (1) [32]

AC C

Position

181.00 153.76

66.46 137.61 129.23 128.71 128.78 180.65 156.98

8 38.10 28.28 73.85 54.95 48.00 21.04 33.09 40.29 48.09 36.30 26.38 122.90 144.43 42.40 31.14 23.68 47.29 42.23 46.39 32.64 36.00 33.44 204.95 10.10 15.81 17.48 23.97 177.47 34.28 23.14 21.11 170.43 66.47 137.61 129.23 128.71 128.78

9 38.36 28.29 77.38 52.68 47.29 20.24 33.11 40.26 48.22 37.06 28.30 123.00 144.45 42.37 31.14 23.71 46.38 42.21 45.73 32.90 36.00 33.45 13.05 16.17 17.51 26.41 177.49 34.29 23.61 157.43 21.40 170.49 66.43 137.61 129.22 128.68 128.76

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Table 4

Antimicrobial activities of gypsogenin (1) and compounds 1a-i as determined by inhibition zone measurements. Inhibition zone (mm) Compounds 1b

1c

1d

1e

1f

1g

1h

1i

Gypsogenin (1)

C30

CN10

TE30

E15

AMP10

NS100

-

-

-

-

-

-

-

-

-

-

24

21

15

11

-

NT

-

-

-

-

-

-

-

-

-

-

17

16

15

8

8

NT

-

-

-

-

-

-

-

-

-

-

25

15

26

30

28

NT

-

-

-

-

-

-

-

-

-

-

23

20

22

23

20

NT

-

-

-

-

-

-

-

-

-

-

22

17

19

11

17

NT

-

-

-

-

-

-

-

-

-

-

21

19

20

14

-

NT

-

-

-

-

-

-

-

-

-

-

22

20

20

21

-

NT

-

-

-

-

-

-

-

-

-

-

17

24

16

20

-

NT

-

-

-

-

-

-

-

-

-

-

16

11

19

-

14

NT

9

9

-

12

14

-

-

-

-

13

23

24

25

26

-

NT

11

11

-

13

14

-

-

-

-

13

10

10

-

12

14

-

-

-

-

13

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

(-): Zone did not occur. NT: Not tested;

RI PT

1a

SC

Escherichia coli ATCC 25922 Salmonella typhimirium ATCC 14028 Micrococcus luteus ATCC 9341 Stapylococcus aureus ATCC 25923 Stapylococcus epidermidis ATCC 12228 Klebsiella pneumoniae ATCC 13882 Pseudomonas aeruginosa ATCC 35032 Proteus vulgaris ATCC 33420 Entereococcus faecalis ATCC 29212 Bacilllus cereus ATCC 11778 Bacillus subtilis ATCC 6633 Bacillus thrungiensis* Candida albicans ATCC 10231 Candida utilis ATCC 9950 Candida trophicalis* Candida glabrata* Saccharomyces cerevisiae ATCC 9763

Reference antibiotics

22

20

12

25

-

NT

26

21

15

28

-

NT

NT

NT

NT

NT

NT

22

NT

NT

NT

NT

NT

21

NT NT

NT NT

NT NT

NT NT

NT NT

15 16

NT

NT

NT

NT

NT

15

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Test Microorganisms

TE D

C30: Chloramphenicol (30 mg, Oxoid); CN10: Gentamycin (10 mg, Oxoid); TE30: Tetracycline (30 mg, Oxoid); E15: Erytromycin (15 mg, Oxoid); AMP10: Ampicillin (10 mg, Oxoid); NS: Nystatin (100 mg, Oxoid); *Special gift from the Faculty of Medicine, Adnan Menderes University.

Table 5

AC C

EP

Compounds 1a, 1b, 1d, 1e, and gypsogenin (1) showed moderate effects against B. cereus ATCC 11778, B. subtilis ATCC 6633, and B. thrungiensis, with compound 1e exhibiting the strongest activity against these strains (inhibition zone = 14 mm) (Table 4). The MIC determination results also showed that these same compounds exhibited moderate antimicrobial activity against the tested microorganisms (Table 5). Again, -1 compound 1e had the highest activity (32 µg mL ), which was similar to that of gypsogenin (1) and more potent than that of streptomycin.

Antimicrobial activities of compounds as determined by MIC values (µg mL-1). Test microorganisms Bacilllus cereus ATCC 11778 Bacillus subtilis ATCC 6633 Bacillus thrungiensis*

1a

1b

1d

1e

Gypsogenin (1)

Str

128

128

64

32

32

64

64

64

32

32

32

64

128

128

64

32

32

64

Compounds 1c, 1f, 1g, 1h, and 1i did not show antibacterial activity. Str = Streptomycin (-) = No effect. * From the Faculty of Medicine, Adnan Menderes University.

2.2.2. Cytotoxic activity The antiproliferative activities of gypsogenin (1) and its derivatives were analyzed by an MTT assay using the following human cancer cell lines in cell culture: HL60 (an acute promyelocytic leukemia cell line), HT-29 and Caco-2 (colorectal adenocarcinoma cell lines), Saos-2 (an osteosarcoma cell line), MCF-7 (a breast cancer cell line), and HeLa (a cervical cancer cell line). The results are shown in Table 6. The IC50 values were determined for each compound and cell line. In addition, the activities of doxorubicin and paclitaxel were determined to serve as comparison compounds. Only the significant (IC50<10 µM) antiproliferative activities were considered for interpreting the compounds as anticancer agents. Gypsogenin (1) showed antiproliferative activity against two cancer cell lines (Saos-2 and MCF-7), with IC50 values less than 10 µM. Interestingly, compounds 1a, 1c, 1d, and 1g were effective against more cell types than gypsogenin (1). The highest activities were found for compounds 1a, 1c, and 1d against HL-60, MCF-7, and HL-60 cell lines, respectively, with IC50 values of 3.90 µM, 5.15 µM, and 5.98 µM, respectively. Thus, these compounds could be possible anticancer agents, with IC50 values comparable to doxorubicin and paclitaxel. Compounds 1b, 1e, 1f, 1h, and 1i were not active against the cell lines tested.

6

ACCEPTED MANUSCRIPT Table 6 Antiproliferative activities of investigated compounds against human cancer cell lines. IC50 1a

1b

1c

1d

1e

1f

1g

1h

1i

Gypsogenin (1)

Doxorubicin

Paclitaxel

HL-60

3.90

10.77

8.14

5.98

12.82

10.62

6.21

6.74

8.68

10.40

0.33

0.32

±1.13

±2.17

±1.44

±1.69

±1.87

±1.86

±1.00

±0.99

±0.67

±2.77

±1.62

±0.79

10.89

11.14

8.51

6.71

13.34

8.01

14.98

10.31

21.43

11.90

0.31

0.29

±2.35

±2.00

±1.11

±0.54

±1.35

±1.63

±2.13

±2.10

±2.56

±1.78

±1.96

±0.66

Caco-2

28.08

17.38

11.06

28.24

30.42

16.13

36.50

51.14

20.28

16.67

0.35

0.31

±2.68

±2.21

±3.26

±2.04

±2.97

±2.73

±3.65

±6.89

±3.08

±3.77

±1.77

±1.12

Saos-2

7.93

8.24

12.70

8.95

10.10

10.28

6.97

10.74

11.70

7.85

0.33

0.35

±1.87

±2.15

±1.72

±1.64

±1.48

±1.97

±0.46

±3.22

±2.46

±1.45

±2.00

±0.93

MCF-7

7.50

20.51

5.15

10.77

7.50

6.67

6.05

65.18

9.90

9.02

0.35

0.33

±2.14

±3.16

±1.18

±2.19

±2.03

±2.64

±1.28

±1.32

±1.33

±2.49

±1.51

±1.53

HeLa

8.71

35.00

74.22

116.23

110.00

28.85

23.62

286.51

40.33

22.48

0.34

0.29

±2.06

±8.70

±15.00

±7.26

±6.45

±2.67

±3.46

±12.18

±1.56

±1.44

HT 29

±2.60

M AN U

Results are expressed as means ± standard deviation.

±4.70

RI PT

Cell line

SC

(µM)

2.2.3. Cell death assay

EP

TE D

The structure-activity relationship was also analyzed. When a different substituent was attached to the gypsogenin aglycone (1), the activity of the compound changed. For example, compound 1a, which has an oxime group on gypsogenin (1), showed higher activity than compound 1b, which contains an acetyl group. In addition, compound 1d (with both an acetyl group and an oxime group) was more active than compound 1f (with both a benzyl group and an oxime group). Compound 1i, which contains oxime, acetyl, and benzyl substituents on the gypsogenin core (1), was not suitable for biological activity, even though compound 1c, which contains a benzyl group, did exhibit antiproliferative activity.

AC C

The apoptotic effects of gypsogenin (1) and compounds 1a-i were determined by the Hoechst 33258 (HO)/propidium iodide (PI) double-staining method. As only the very late phenotypes of apoptosis can be visualized by phase contrast microscopy, the quantification of cell death impossible using microscopy alone. However, the combination of microscopic examination together with a double-staining method with fluorescent dyes is both a reliable and sensitive method to determine the type of cell death and to quantify it. HO and PI both stain chromatin. When HO/PI-stained cells are examined under a microscope, the early stages of death are also detected and can be quantified. In combination with the examination of condensed chromatin, this method allows discrimination between early or late apoptosis, necrosis, and intact cells because administration of both dyes either stains the nuclei blue when the cells are viable or in an early-

to mid-apoptotic stage, or pink (interference of blue and red) when the cells are in a late apoptotic stage or are necrotic. Thus, HO/PI staining facilitates distinguishing between apoptosis and necrosis. The highest apoptotic rates were determined with gypsogenin (1) and compound 1a. At 48 h after the application of these compounds, between 20% and 36% of the cells died through an apoptotic pathway in the different cell lines tested. Paclitaxel, a cytoskeletal drug that targets tubulin and stabilizes the microtubule polymer, was used as a positive control. This drug blocks the progression of mitosis, thus triggering the apoptotic pathway at the mitotic checkpoint. Paclitaxel showed approximately 50% apoptosis at a dose of 0.5 µM. In this study, only gypsogenin (1) and compound 1a approached these levels, with 20–36% apoptosis achieved at a concentration of 20 µM and very low necrosis rates (Table 7). Thus, gypsogenin (1) and compound 1a triggered the apoptotic pathway in cancer cell lines. Normally, approximately 50% of cancers have their apoptotic cell death mechanism disabled. Therefore, this endpoint is an important approach to evaluate the effects of such compounds. In contrast, compound 1b was found to have necrotic effects with a high cell death value (20–55%) in all tested cell lines. Compounds 1c, 1f, and 1i could be described as apoptotic/necrotic agents, but the necrotic ratios were higher, causing cell death at high levels. Compounds 1f and 1i were obtained from compound 1c, implying that the activities of compounds 1f and 1i are related to those of compound 1c. Meanwhile, compounds 1d, 1e, 1g, and 1h showed mostly necrotic effects in all cell lines. Only a few apoptotic cells were observed by microscopic observations.

7

ACCEPTED MANUSCRIPT Table 7 Apoptosis and necrosis ratios in different cell lines after the application of 20 µM test compound or 0.5 µM control.

Cell line

1a

1b

1c

1d

1e

Apoptosis/Necrosis (%) 1f 1g 1h (20 µM)

1i

Gypsogenin (1)

Doxorubicin Paclitaxel (0.5 µM)

30/3

8/25

6/48

6/52

2/67

8/41

2/64

3/51

5/50

33/4

2/45

27/0.7

0.8/20

1/22

0.6/28

3/26

12/29

2/32

0.7/29

24/14

21/2

5/49

53/0.0 52/2

Caco

23/3

3/55

1/15

0.0/17

3/29

11/32

4/48

1/37

19/33

20/0.0

4/47

52/2

2/34

42/1

Saos

27/3

3/44

9/30

1/46

2/61

9/23

5/41

0.0/45

24/44

36/2

MCF-7

26/4

5/29

4/57

5/55

6/63

12/54

4/63

5/36

10/48

HeLa

26/3

4/33

4/45

0.0/64

2/65

11/32

5/55

2/68

15/50

RI PT

HL-60 HT-29

50/1

29/0.0

2/28

44/2

LC-MS was recorded on an AGILENT 1200 Capillary spectrometer. Column chromatography was carried out using 60 Å silica gel (Merck 7734). TLC was carried out using 60 Å silica gel on F254 aluminum plates (Merck 5554).

AC C

EP

TE D

M AN U

In summary, we synthesized nine gypsogenin derivatives in good yield. Gypsogenin (1) and its derivatives were evaluated for their antimicrobial (against twelve bacteria and five yeasts) and cytotoxic activities. Furthermore, gypsogenin (1) and compounds 1a, 1b, 1d, and 1e were assessed against three Bacillus species using the MIC method. Among all of these compounds, 1a, 1b, 1d, and 1e as well as gypsogenin (1) showed activities only against Gram-positive bacteria (B. cereus ATCC 11778, B. subtilis ATCC 6633, and B. thrungiensis). According to our results, compounds 1d and 1e had stronger effects against B. subtilis ATCC 6633 compared to the other compounds. In addition, compound 1e and gypsogenin (1) were more potent against B. cereus ATCC 11778, B. subtilis ATCC 6633, and B. thrungiensis, compared to the other compounds. However, none of the compounds showed antifungal effects. The synthesized compounds were tested against six human cancer cell lines, and compounds 1a, 1c, and 1d could be considered as possible anticancer agents as they were shown to affect the cell cycle or check points, causing cell cycle arrest and cell death. In particular, gypsogenin (1) and compound 1a triggered the apoptotic pathway in cancer cells, showing high apoptosis ratios. As cell cycle arrest and apoptosis are significant endpoints/targets in cancer therapy, these novel gypsogenin derivatives have potential as anticancer therapeutics.

1/44

SC

3. Conclusion

30/4

4. Experimental 4.1. General Melting points of all compounds were recorded on a Gallenkamp electrothermal melting point apparatus and are uncorrected. IR spectra were recorded on a PerkinElmer Spectrum 100 FT-IR spectrometer. NMR spectra 1 were measured in pyridine-d5 at 400 MHz for H NMR, HMBC, HMQC, and NOESY experiments and at 100 13 MHz for C NMR on a Varian AS-400 spectrometer.

4.2. General method for the preparation of gypsogenin (1) The commercially available water extract of G. arrostii roots (400 mL) was mixed with ethanol (100 mL) and hydrolyzed with 10% KOH at 100 °C for two days. The reaction mixture was collected and neutralized with HCl. Then, this mixture was hydrolyzed with 10% HCl for three days before being neutralized with KOH and extracted with CH2Cl2. The CH2Cl2 phase was concentrated to give 1,4504 g of crude gypsogenin (1). The crude product was purified by column chromatography with hexane/ethyl acetate (6/4) as the eluent to afford gypsogenin (1) (325 mg) [32].

4.2.1. 3-Hydroxy-23-oxoolean-12-en-28-oic acid (gypsogenin (1)) mp: 273–274 °C; LC/MS (ESI-MS) m/z = 469.20 (M-1) (negative ion mode).

4.3. General procedure for the synthesis of compounds 1a, 1d, 1f, and 1i -4

Gypsogenin (1) (100 mg, 2.13×10 mol) and -4 compounds 1b (60 mg, 1.17×10 mol), 1c (75 mg, -4 -5 1.34×10 mol), and 1h (60 mg, 9.97×10 mol) were mixed with hydroxylamine hydrochloride (NH2OH·HCl) (6 mg, 20 mg, 30 mg, 13 mg, respectively) and sodium acetate and dissolved in acetonitrile (6 mL) and water (2 mL). The reaction mixture was stirred at room temperature for 24 h. After acetonitrile evaporation, water (10 mL) was added to this mixture, and the mixture was extracted with CH2Cl2 (3×15 mL). The organic layer was dried over anhydrous sodium sulfate, filtered, and concentrated in vacuo. The residue was purified by column chromatography using hexane/ethyl

8

ACCEPTED MANUSCRIPT 4.4. General procedure for the synthesis of compounds

4.4.1. 3-(Acetyloxy)-23-oxoolean-12-en-28-oic acid (1b) -4 Prepared gypsogenin (1) (125 mg, 2.66×10 mol), pyridine (1 mL), and acetic anhydride were mixed according to the above general procedure to provide 133 mg of compound 1b as a white solid. Yield: 97.8%; m.p. 164.8–166.1 °C; UV λmax (CH2Cl2): 208.0 nm; IR -1 νmax (CH2Cl2) cm : 3848, 3573, 3353, 2946, 1736, 1694, 1463, 1370, 1238, 1030, 1009, 736, 688, 645, 603, 509, 485; LC/MS (ESI-MS) m/z = 511.20 (M-1) (negative ion mode).

M AN U

4.3.2. 3-(Acetyloxy)-23-(hydroxyimino)olean-12-en-28oic acid (1d) -4 Prepared compound 1b (60 mg, 1.17×10 mol), hydroxylamine hydrochloride (NH2OH·HCl) (20 mg, -4 -4 2.88×10 mol), and sodium acetate (40 mg, 4.87×10 mol) were mixed according to the above general procedure to provide 60 mg of compound 1d as a light yellow solid. Yield: 97.2%; m.p. 251.4–253.0 °C; UV -1 λmax (CH2Cl2): 209.0 nm; IR νmax (CH2Cl2) cm : 3817, 3649, 3254, 2950, 2639, 1730, 1689, 1459, 1369, 1265, 1242, 1032, 958, 818, 736, 690, 645, 568; LC/MS (ESIMS) m/z = 526.20 (M-1) (negative ion mode).

Acetic anhydride (5 mL) was added to gypsogenin -4 (1) (125 mg, 2.66×10 mol) in pyridine (1 mL) and to -4 compound 1c (100 mg, 1.79×10 mol) in pyridine (2 mL). The mixture was allowed to stir for 48 h at room temperature. After stirring, the mixture was extracted with CH2Cl2 (3×10 mL). The organic layer was dried over anhydrous sodium sulfate and evaporated to dryness. The residue was purified by silica gel chromatography using hexane/ethyl acetate (6/4) to give compounds 1b and 1h, respectively.

RI PT

4.3.1. 3-Hydroxy-23-(hydroxyimino)olean-12-en-28-oic acid (1a) -4 Prepared gypsogenin (1) (100 mg, 2.13×10 mol), hydroxylamine hydrochloride (NH2OH·HCl) (6 mg, -5 -4 8.63×10 mol), and sodium acetate (13.7 mg, 1.67×10 mol) were mixed according to the above general procedure to provide 72.3 mg of compound 1a as a white solid. Yield: 70.2%; m.p. 268.1–270.0 °C; UV λmax -1 (CH2Cl2): 205.0 nm; IR νmax (CH2Cl2) cm : 3912, 3375, 2854, 1686, 1463, 1363, 1273, 957, 817, 647, 520, 475; LC/MS (ESI-MS) m/z = 484.20 (M-1) (negative ion mode).

1b and 1h

SC

acetate (6/4) to give compounds 1a, 1d, 1f, and 1i, respectively.

AC C

EP

TE D

4.3.3. Benzyl 3-hydroxy-23-(hydroxyimino)olean-12-en28-oate (1f) -4 Prepared compound 1c (75 mg, 1.34×10 mol), hydroxylamine hydrochloride (NH2OH·HCl) (30 mg, -4 -4 4.32×10 mol), and sodium acetate (60 mg, 7.31×10 mol) were mixed according to the above general procedure to provide 68 mg of compound 1f as a light yellow solid. Yield: 88.3%; m.p. 139.4–141.0 °C; UV -1 λmax (CH2Cl2): 209.0 nm; IR νmax (CH2Cl2) cm : 3806, 3300, 2945, 2590, 2297, 1738, 1723, 1456, 1385, 1365, 1261, 1230, 1216, 1159, 1121, 1084, 1030, 934, 819, 736, 695, 529, 463; LC/MS (ESI-MS) m/z = 576.40 (M+1) (positive ion mode).

4.3.4. Benzyl 3-(acetyloxy)-23-(hydroxyimino)olean-12en-28-oate (1i) -5 Prepared compound 1h (60 mg, 9.97×10 mol), hydroxylamine hydrochloride (NH2OH·HCl) (13 mg, -4 -4 1.87×10 mol), and sodium acetate (45 mg, 5.49×10 mol) were mixed according to the above general procedure to provide 56 mg of compound 1i as a white solid. Yield: 91.2%; m.p. 224.5–226.0 °C; UV λmax -1 (CH2Cl2): 205.0, 290.0 nm; IR νmax (CH2Cl2) cm : 3696, 3350, 2920, 2850, 2130, 1696, 1635, 1534, 1463, 1386, 1364, 1278, 1182, 1016, 956, 720, 601, 529, 464, 457; LC/MS (ESI-MS) m/z = 618.40 (M+1) (positive ion mode).

4.4.2. Benzyl 3-(acetyloxy)-23-oxoolean-12-en-28-oate (1h) -4 Prepared compound 1c (100 mg, 1.79×10 mol), pyridine (2 mL), and acetic anhydride were mixed according to the above general procedure to provide 105 mg of compound 1h as a white solid. Yield: 97.7%; m.p. 159.6–161.1 °C; UV λmax (CH2Cl2): 208.0, 290.0 -1 nm; IR νmax (CH2Cl2) cm : 3839, 3736, 3100, 2947, 2876, 1733, 1686, 1458, 1369, 1238, 1159, 1121, 1030, 1010, 976, 897, 736, 647, 604, 558; LC/MS (ESI-MS) m/z = 603.40 (M+1) (positive ion mode).

4.5. General procedure for the synthesis of compound 1c -4

A mixture of gypsogenin (1) (400 mg, 8.51×10 ) and triethylamine (3 mL) was stirred at room temperature for 1 h before benzyl bromide (2 mL) was added. The mixture was refluxed for 3 h and then stirred at room temperature for 48 h. Water (10 mL) was added to the reaction mixture, and the mixture was extracted with CH2Cl2 (3×10 mL). The organic layer was dried over anhydrous sodium sulfate and evaporated to dryness. The residue was purified by column chromatography on silica gel using hexane/ethyl acetate (4/6) to give compound 1c as an amorphous powder (260 mg). 4.5.1. Benzyl 3-hydroxy-23-oxoolean-12-en-28-oate (1c) Yield: 54.6%; UV λmax (CH2Cl2): 207.0 nm; IR νmax -1 (CH2Cl2) cm : 3290, 2964, 2855, 2700, 1750, 1466, 1458, 1383, 1358, 1317, 1258, 1217, 1158, 1117, 1075, 1042, 1025, 958, 833, 733, 683, 608, 542; LC/MS (ESIMS) m/z = 561.40 (M+1) (positive ion mode).

9

ACCEPTED MANUSCRIPT The antibacterial and antifungal activities of

The antimicrobial activities were determined according to the standard Antimicrobial Disc Susceptibility Tests summarized by the National Committee for Clinical Laboratory Standards [37]. Fresh -1 stock solutions (1000 µg mL ) of all the synthesized compounds were prepared in DMSO according to the desired concentrations for the experiments. The inoculum suspensions of the tested bacteria and yeasts were prepared from the broth cultures (18–24 h), and the turbidity was adjusted to be equivalent to a 0.5 McFarland standard tube to give a concentration of 8 6 -1 1×10 bacterial cells and 1×10 yeast cells mL , respectively. To test the antimicrobial activity of all the synthesized compounds, a Mueller Hinton Agar (MHA) plate was inoculated with 0.1 mL of broth culture of bacteria or yeast. Then, a hole of 6 mm in diameter and depth was made on top of the agar with a sterile stick and filled with 30 µL of each synthesized compound, respectively. Plates inoculated with E. coli, S. typhimurium, S. aureus, S. epidermidis, E. faecalis, K. pneumonia, P. aeruginosa, and P. vulgaris were incubated at 37 °C for 24 h, while those with M. luteus, B. subtilis, B. cereus, B. thrungiensis, S. cerevisiae, C. albicans, C. utilis, C. glabrata, and C. trophicalis were incubated at 30 °C for 24 h. At the end of the incubation time, the diameters of the inhibition zones formed on the MHA were evaluated in millimeters. Discs of chloramphenicol (C30), gentamycine (CN10), tetracycline (TE30), erytromycine (E15), ampicillin (AMP10), and nystatine (NS100) were used as positive controls. The measured inhibition zones of the study compounds were compared with those of the reference discs.

M AN U

4.6.1. 3-(Acetyloxy)-23-[(aminocarbonothioyl) hydrazono]olean-12-en-28-oic acid (1e) -4 Prepared compound 1b (125 mg, 2.44×10 ) and -3 thiosemicarbazide (120 mg, 1.32×10 mol) were mixed according to the above general procedure to provide 65 mg of compound 1e as a light yellow solid. Yield: 45.8%; m.p. 205.3–207.4 °C; UV λmax (CH2Cl2): 211.0, -1 261.0 nm; IR νmax (CH2Cl2) cm : 3854, 3629, 3434, 3267, 3164, 2950, 1734, 1691, 1597, 1534, 1459, 1366, 1274, 1260, 1245, 1088, 1028, 1008, 1055, 839, 820, 764, 749, 642, 530, 462; LC/MS (ESI-MS) m/z = 584.20 (M-1) (negative ion mode).

4.7.1.1. Disc diffusion method

RI PT

A solution of thiosemicarbazide in water (7 mL) was added to a solution of compounds 1b (125 mg, -4 -4 2.44×10 ) and 1c (87 mg, 1.55×10 ) in MeOH (7 mL). The mixture was refluxed for 13 h. After cooling, the mixture was extracted with CH2Cl2 (3×10 mL). The organic layer was dried over anhydrous sodium sulfate and evaporated to dryness. The residue was purified by column chromatography on silica gel using hexane/ethyl acetate (6/4) to give compounds 1e and 1g, respectively.

all nine synthesized compounds were analyzed by the disc diffusion method and the broth dilution method to determine the MIC.

SC

4.6. General procedure for the synthesis of compounds 1e and 1g

AC C

4.7. Pharmacology

EP

TE D

4.6.2. Benzyl 23-[(aminocarbonothioyl)hydrazono]-3hydroxyolean-12-en-28-oate (1g) -4 Prepared compound 1c (87 mg, 1.55×10 ) and -4 thiosemicarbazide (50 mg, 5.48×10 mol) were mixed according to the above general procedure to provide 56 mg of compound 1g as a white solid. Yield: 56.9%; m.p. 138.1–139.0 °C; UV λmax (CH2Cl2): 207.0; 270.0 nm; IR -1 νmax (CH2Cl2) cm : 3901, 3819, 3567, 3244, 2970, 2300, 1743, 1593, 1531, 1365, 1276, 1229, 1216, 1158, 1082, 958, 832, 749, 662, 529, 474, 464, 457; LC/MS (ESI-MS) m/z = 634.40 (M+1) (positive ion mode).

4.7.1. Antibacterial and antifungal activity All of the compounds used in this study were tested for their in vitro antibacterial and antifungal effects (antimicrobial activity) following standard methods (Supplementary data, Figure 2). In this study, twelve bacterial cultures and five different yeasts were used. Eleven bacterial strains and three yeast strains were obtained from the American Type Culture Collection (ATCC; Rockville, MD, USA). The other bacterial strain, B. thrungiensis, was isolated from human fecal samples obtained from Adnan Menderes University (Clinical Microbiology Laboratory, Faculty of Medicine) and was cultured in Nutrient Broth (Merck) at 30 °C for 24 h. The other two yeast strains, C. glabrata and C. trophicalis, were cultured in Malt Extract Broth (Merck, USA) at 30 °C for 24 h.

4.7.1.2. Dilution method Screening for antibacterial activities was carried out by preparing a microdilution broth, following the procedure outlined in the Manual of Clinical Microbiology [38]. All the bacteria were inoculated in the nutrient broth and incubated at 30 °C for 24 h. The -1 compounds were dissolved in DMSO (2 mg mL ) and then diluted in Mueller Hinton broth. Two-fold serial dilutions of the compounds were employed to determine -1 the MIC, ranging from 256 to 0.125 µg mL . Cultures were grown at 30 °C for 18–20 h, and the final inoc ulum 6 -1 was approximately 10 cfu mL . Test cultures were incubated at 37 °C for 24 h. The lowest concentrati on of antimicrobial agent that resulted in complete inhibition of the microorganisms was represented as the MIC (µg -1 mL ). Streptomycin (I.E. Ulagay) was used as a positive control in the dilution method.

10

ACCEPTED MANUSCRIPT Appendix A. Supplementary data 4.7.2. Cell culture

[1]

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Cells were seeded in 48-well plates at a 4 5 -1 concentration of 1×10 –1×10 cells mL and incubated with increasing concentrations of agents (corresponding to 0.5, 10, 20, and 40 µM of the compounds or controls). Cell viability was determined at 24 and 72 h with the MTT assay carried out with a Thermo Scientific Multiskan spectrometer. The absorbance at 590 nm was recorded using Thermo Scientific SkanIt Software. The IC50 values were analyzed using GraphPad Prism 5.0 software. Experiments were done in triplicate. The percentage of viable cells was compared to the untreated control and calculated as follows: [(C72 h + drug - C24 h + drug) / (C72 h - drug - C24 h - drug)] × 100 = % cell division, where C72 h + drug is the cell number after drug treatment for 72 h, C24 h + drug is the cell number after drug treatment for 24 h, C72 h drug is the cell number after 72 h without drug treatment, and C24 h - drug, is the cell number after 24 h without drug treatment.

References

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4.7.3. Cytotoxicity testing

Supplementary data related to this article can be found at http://

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HL-60, HT-29, Caco-2, Saos-2, MCF-7, and HeLa cell lines were purchased from ATCC. Growth media and its supplements were purchased from Life Science Tech. (Sacem, Turkey). Cells were grown in RPMI1540, Dulbecco’s modified Eagle’s medium, or McCoys 5A medium supplemented with 10–15% heat inactivated fetal calf serum, 1% L-glutamine, 1% penicillin/streptomycin, and nonessential amino acids and cultivated at 37 °C in a humidified atmosphere containing 5% CO2. Control chemicals doxorubicin and paclitaxel were purchased from Cell Signalling Tech., Inc. (Beverly, MA, USA).

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4.7.4. Hoechst 33258 (HO) and propidium iodide (PI) double staining

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Cells were seeded in 48-well plates and incubated with increasing concentrations of agents (corresponding to 0.5, 10, 20, and 40 µM of the compounds or controls) for 24 and 48 h (Supplementary data, Figure 3). After microscopic examination of the coloured cells and considering the IC50 values, it was decided to use the effect obtained with a compound concentration of 20 µM in the cell death assay. The effective time point was defined as 48 h. Cell death quantification was performed according to a previously described method [39]. For microscopic analyses, an Olympus BX52 fluorescence microscope was used, and the fluorescent dyes HO and PI were purchased from Sigma. Acknowledgements We are grateful to Professor Dr. Hüseyin Anıl for his valuable suggestions for the synthetic routes.

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F. Melzig, Triterpenoid saponins of the Caryophyllaceae and Illecebraceae family, Phytochemistry Letters 4 (2011) 59-68. Y. C. Kim, R. Higuchi, T. Komori, Application of hydrothermolysis to the studies on constituents of the Merck saponin, Liebigs Annalen der Chemie (1992) 941-946. S. Emirdağ-Öztürk, İ. Babahan, A. Özmen, Synthesis, Characterization and in vitro anti-neoplastic activity of Gypsogenin Derivatives, Bioorganic Chemistry 53 (2014) 15-23. http://dx.doi.org/10.1016/j.bioorg.2013.12.001 Y. Liu, W-X. Lu, M-C. Yan, Y. Yu, T. Ikejima, M-S Cheng, Synthesis and Tumor Cytotoxicity of Novel Amide Derivatives of β-Hederin, Molecules 15 (2010) 7871-7883. M.-C. Yan, Y. Liu, H. Chen, Y. Ke, Q.-C. Xu, M.-S. Cheng, Synthesis and antitumor activity of two natural Nacetylglucosamine-bearing triterpenoid saponins: Lotoidoside D and E, Bioorganic & Medicinal Chemistry Letters 16 (2006) 4200-4204. M.-S. Cheng, M.-C. Yan, Y. Liu, L.-G. Zheng, J. Liu, Synthesis of βhederin and Hederacolchiside A1: triterpenoid triterpenoid saponins bearing a unique cytotoxicity-inducing disaccharide moiety, Carbohydrate Research 341 (2006) 60–67. C. H. Collins, P. M. Lyne, J. M. Grange, Collins and Lyne's Microbiological Methods, Seventh ed. Butterworth Heinemann, London, UK Chap. 8 (1995) 121–136. National Committee for Clinical Laboratory Standards (1993). Performance Standards for Antimicrobial Disk Susceptibility Tests, Approved Standard NCCLS Publication, Villanova, PA, USA, M2A51-32. P. R. Murray, E. J. Baron, M. A. Pfaller, F. C. Tenover, R. H. Yolke, Manual of Clinic Microbiology, 7. ed. ASM Press D. C., Washington (1995) 1773. M. Grusch, D. Polgar, S. Gfatter, K. Leuhuber, S. Huettenbrenner, C. Leisser, G. Fuhrmann, F. Kassie, H. Steinkellner, K. Smid, G. J. Peters, H. Jayaram, W. Klepal, T. Szekeres, S. Knasmuller, G. Krupitza, Maintenance of ATP favours apoptosis over necrosis triggered by benzamide riboside, Cell Death Differentiation 9 (2) (2002) 169–178.

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C. Lavaud, L. Voutquenne, G. Massiot, L. L. Men-Olivier, B. C. Das, O. Laprevote, L. Serani, C. Delaude, M. Becchi, Saponins from the stem bark of Filicium decipiens, Phytochemistry 47 (1998) 441-449. J.-G. Luo, W. Nie, L.-Y. Kong, Three new sulfated triterpenoids from the roots of Gypsophila pacifica, Journal of Asian Natural Products Research 13 (2011) 529-533. I. Arslan, A. Celik, M. F. Melzig, Nebulosides A-B, novel triterpene saponins from under-ground parts of Gypsophila arrostii Guss. var. nebulosa, Bioorganic & Medicinal Chemistry 21 (2013) 1279-1283. W. Nie, J.G. Luo, L. Y. Kong, New triterpenoid saponins from the roots of Gypsophila pacifica Kom, Carbohydrate Research 345 (2010) 68-73. M.-K. Sung, C. W. C. Kendall, A. V. Rao, Effect of soybean saponins and gypsophila saponin on morphology of colon carcinoma cells in culture, Food and Chemical Toxicology 33 (1995) 357-366. D. J. Marciani, Triterpene saponin analogs having adjuvant and immunostimulatory activity, U.S. Patent 5,977,081 (1999). L. Voutquenne-Nazabadioko, R. Gevrenova, N. Borie, D. Harakat, C. Sayagh, A. Weng, M. Thakur, M. Zaharieva, M. Henry, Triterpenoid saponins from the roots of Gypsophila trichotoma Wender, Phytochemistry 90 (2013) 114-127. J.-G. Luo, L. Ma, L.-Y. Kong, New triterpenoid saponins with strong α-glucosidase inhibitory activity from the roots of Gypsophila oldhamiana, Bioorganic & Medicinal Chemistry 16 (2008) 29122920. R. Gevrenova, T. Stancheva, Y. Voynikov, D. Laurain-Mattar, M. Henry, Root in vitro cultures of six Gypsophila species and their saponin contents, Enzyme and Microbial Technology 47 (2010) 97104. H. Bai, Y. Zhong, Y.-Y. Xie, Y.-S. Wang, L. Liu, L. Zhou, J. Wang, Y.L. Mu, C.-X. Zuo, A Major Triterpenoid Saponin from Gypsophila oldhamiana, Chemistry & Biodiversity 4 (2007) 955-960. M. Yotova, I. Krasteva, K. Jenett-Siems, P. Zdraveva, S. Nikolov, Triterpenoids in Gypsophila trichotoma Wend, Phytochemistry Letters 5 (2012) 752-755.

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Highlights Nine novel gypsogenin derivatives (1a-i) were semisynthesized.

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1b, 1d, 1e showed the best antimicrobial activities against Bacillus cereus.

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1a 3.9 µM, 1c 5.15 µM, 1d 5.98 µM showed remarkable cytotoxic activity.

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Gypsogenin and 1a triggered the apoptotic mechanism at a concentration of 20 µM.

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Figure 1: 1H NMR and 13C NMR, ESI mass spectrum and IR spectra for compound 1a-i. 1

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H NMR spectrum of compound 1a recorded on a Varian AS-400 spectrometer (400 MHz, pyridine-d5):

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C NMR spectrum of compound 1a recorded on a Varian AS-400 spectrometer (100 MHz, pyridine-d5):

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ESI mass spectrum of compound 1a recorded on an AGILENT 1200 Capillary spectrometer:

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For compound 1a, IR spectra were recorded on a Perkin-Elmer Spectrum 100 FT-IR spectrometer:

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H NMR spectrum of compound 1b recorded on a Varian AS-400 spectrometer (400 MHz, pyridine-d5):

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C NMR spectrum of compound 1b recorded on a Varian AS-400 spectrometer (100 MHz, pyridine-d5):

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ESI mass spectrum of compound 1b recorded on an AGILENT 1200 Capillary spectrometer:

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-MS, 9.7-12.6min 511.2

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For compound 1b, IR spectra were recorded on a Perkin-Elmer Spectrum 100 FT-IR spectrometer:

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H NMR spectrum of compound 1c recorded on a Varian AS-400 spectrometer (400 MHz, pyridine-d5):

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C NMR spectrum of compound 1c recorded on a Varian AS-400 spectrometer (100 MHz, pyridine-d5):

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ESI mass spectrum of compound 1c recorded on an AGILENT 1200 Capillary spectrometer:

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For compound 1c, IR spectra were recorded on a Perkin-Elmer Spectrum 100 FT-IR spectrometer:

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H NMR spectrum of compound 1d recorded on a Varian AS-400 spectrometer (400 MHz, pyridine-d5):

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C NMR spectrum of compound 1d recorded on a Varian AS-400 spectrometer (100 MHz, pyridine-d5):

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ESI mass spectrum of compound 1d recorded on an AGILENT 1200 Capillary spectrometer:

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For compound 1d, IR spectra were recorded on a Perkin-Elmer Spectrum 100 FT-IR spectrometer:

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H NMR spectrum of compound 1e recorded on a Varian AS-400 spectrometer (400 MHz, pyridine-d5):

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C NMR spectrum of compound 1e recorded on a Varian AS-400 spectrometer (100 MHz, pyridine-d5):

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ESI mass spectrum of compound 1e recorded on an AGILENT 1200 Capillary spectrometer:

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For compound 1e, IR spectra were recorded on a Perkin-Elmer Spectrum 100 FT-IR spectrometer:

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H NMR spectrum of compound 1f recorded on a Varian AS-400 spectrometer (400 MHz, pyridine-d5):

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C NMR spectrum of compound 1f recorded on a Varian AS-400 spectrometer (100 MHz, pyridine-d5):

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ESI mass spectrum of compound 1f recorded on an AGILENT 1200 Capillary spectrometer:

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For compound 1f, IR spectra were recorded on a Perkin-Elmer Spectrum 100 FT-IR spectrometer:

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H NMR spectrum of compound 1g recorded on a Varian AS-400 spectrometer (400 MHz, pyridine-d5):

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C NMR spectrum of compound 1g recorded on a Varian AS-400 spectrometer (100 MHz, pyridine-d5):

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ESI mass spectrum of compound 1g recorded on an AGILENT 1200 Capillary spectrometer:

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H NMR spectrum of compound 1h recorded on a Varian AS-400 spectrometer (400 MHz, pyridine-d5):

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C NMR spectrum of compound 1h recorded on a Varian AS-400 spectrometer (100 MHz, pyridine-d5):

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ESI mass spectrum of compound 1h recorded on an AGILENT 1200 Capillary spectrometer:

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H NMR spectrum of compound 1i recorded on a Varian AS-400 spectrometer (400 MHz, pyridine-d5):

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C NMR spectrum of compound 1i recorded on a Varian AS-400 spectrometer (100 MHz, pyridine-d5):

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ESI mass spectrum of compound 1i recorded on an AGILENT 1200 Capillary spectrometer:

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Figure 2: Photographies (effect of against B. thrungiensis, B. cereus, Bacillus subtilis) for gypsogenin (1) and compound 1a, 1b, 1d, 1e.

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Bacillus thrungiensis.( 1a, 1b, 1d and 1e)

Photography 1: 13.22.32: Effects of compound 1a, 1b, 1d and 1e against Bacillus thrungiensis.

Bacillus thrungiensis (Gypsogenin= sentral of petri dish)

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Photography 2: 13.23.03: Effects of gypsogenin (1) against Bacillus thrungiensis.

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Bacillus cereus ATCC 11778

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(1a, 1b, 1d and 1e)

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Photography 3: 13.23.25: Effects of compound 1a, 1b, 1d, 1e against Bacillus cereus ATCC 11778.

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Bacillus cereus ATCC 11778

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Photography 4: 13.23.39: Effects of gypsogenin (1) against Bacillus cereus ATCC 11778.

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Bacillus subtilis ATCC 6633

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Photography 5: 13.24.04: Effects of compounds 1a, 1b, 1d and 1e against Bacillus subtilis ATCC 6633.

Bacillus subtilis ATCC 6633 (Gypsogenin (1)= sentral of petri dish)

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Photography 6: 13.23.54: Effects of gypsogenin against Bacillus subtilis ATCC 6633.

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Figure 3:

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Figure 3.1. Early and late apoptotic cells observed by HO/PI double staining (48 h, HL60 cell line, paclitaxel 0,5 µM ). Photographed by Ali ÖZMEN-2014

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Figure 3.2. Presentation of the cytotoxic effects and apoptotic/necrotic ratios of gypsogenin (1), compound 1a, compound 1c and compound 1d in four different cell lines which they found effective against. These compounds were selected by considering their cytotoxic effect and cell death ratios together. The best results were obtained in applied substances (chemicals) at 20 µM and in control chemicals 0,5 µM. Therefore in these whole graphs only the data from the selected cell lines, substances (chemicals) and effective concentrations has been presented.

b- Results against HT-29 cell line: Among the selected compounds; compound 1d has shown high cytotoxicity at 20 µM concentration. There is a significant increase in necrotic cells after 48 h application of compound 1d. Gypsogenin (1) itself was also effective against HT-29 cell line and causes apoptotic cell death.

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a- Results against HL-60 cell line: Among the selected compounds; compound 1a and compound 1d have shown high cytotoxicity at 20 µM concentration. Compound 1a and 1d were find to be different by exhibiting their cytotoxicity. There is a significant increase in apoptotic cells after 48 h application of compound 1a. Conversely compound 1d causes necrotic cell deaths. Gypsogenin itself was also effective against HL-60 cell line and causes apoptotic cell death.

c- Results against MCF-7 cell line: Among the selected compounds; compound 1c has shown very high cytotoxicity at 20 µM concentration. There is a significant increase in necrotic cells after 48 h application of compound 1c with low ratio of apoptotic cells. The same effects have been observed with compound 1d. Gypsogenin (1) itself was also effective against MCF-7 cell line and causes a very high level of apoptotic cell death.

d- Results against Saos-2 cell line: Among the selected compounds; gypsogenin (1) has revealed the best effect at 20 µM concentration with high apoptotic cells and a few necrotic. In this cell line all the compounds were not so effective such as the other tested cell lines. There is a weak effect on cytotoxicity with compound 1d but nearly all of the died cells were found to be necrotic.